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Reactive Heterocycles for Examining Polyketide BiosynthesisPrasad, Gitanjeli 01 September 2013 (has links)
Polyketides are a class of natural products that exhibit remarkable structural and functionally diversity and are highly sought after due to their medicinally important activities. For many decades now, polyketide synthases (PKSs), the mega-enzymes responsible for biosynthesis of polyketides have been the focus of extensive investigation to make new polyketides by polyketide engineering strategies. While there are many established methods to investigate polyketide enzymes and biosynthesis mechanisms, they have substantial shortcomings that have limited the extent of success with polyketide engineering efforts.
This thesis focuses on developing simple, flexible yet powerful tools for examining polyketide biosynthesis by overcoming some deficiencies in currently used techniques. Reactive heterocylces have been designed for direct labeling of key polyketide synthase enzymes to provide a direct insight into its functions and mechanisms. First β-lactones and then β-lactams have been used as small molecule probes to perform site-specific labeling of acyl carrier proteins and further used for mechanistic interrogation of key steps in polyketide biosynthesis. The utility of these probes has been demonstrated by comparison to traditional probes and has been successfully applied to examine substrate selectivity of keto synthases, key enzymes in polyketide biosynthesis. The applications of the tools described in this manuscript only scratch the surface of their capabilities and are expected to significantly aid in the study of new and existing PKS systems leading to improved understanding of how these extraordinary biosynthetic machines function.
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Site-specific labeling of affinity molecules for in vitro and in vivo studiesPerols, Anna January 2014 (has links)
The thesis is focused on site-specific labeling of affinity molecules for different applications where two types of binding proteins, Affibody molecules and antibodies, have been used. For the purpose of improving the properties of Affibody molecules for in vivo imaging, novel bi-functional chelators for radiolabeling using the radionuclide 111In were evaluated. In a first study, two chelators denoted NOTA and DOTA, respectively, were separately conjugated via maleimide chemistry to a C-terminal cysteine residue in a HER2-binding Affibody molecule (ZHER2:2395). In vivo evaluation using mice with prostate carcinoma cell line xenografts showed that the 111In-NOTA-MMA-ZHER2:2395 tracer exhibited faster clearance from blood than the 111In-DOTA-MMA-ZHER2:2395 counterpart,resulting in improved tumor-to-organ ratios. In a second study the in vivo imaging properties of a third tracer, 111In-NODAGA-MMA-ZHER2:2395, was investigated in tumor-bearing mice. While the tumor uptake was lower than seen for the 111In-DOTA-MMA-ZHER2:2395 tracer, a low uptake in non-targeted organs and a fast clearance from blood resulted in higher tumor-to-organ ratios for 111In-NODAGA-MMA-ZHER2:2395 compared to the DOTA variant. In a following study, a synthetically produced HER2-targeting affibody variant, denoted ZHER2:S1, was used where NODAGA, NOTA and DOTA chelators instead were conjugated via an amide bond to the N-terminus. In vivo evaluation in mice showed an unfavorable uptake in liver for 111In-NOTA-ZHER2:S1, resulting in a discontinuation. The study showed faster clearance of 111In-NODAGA-ZHER2:S1 from blood, but also an increased uptake in bone in comparison to 111In-DOTA-ZHER2:S1. As bone is a common metastatic site in prostate cancer, the favorable tumor-to-bone ratio for 111In-DOTA-ZHER2:S1 suggests it as the tracer of choice for prostate cancer. Further, the DOTA chelator was also evaluated as conjugated to either N- or C-terminus or to the back of helix 3 via an amide bond, where the in vivo evaluation showed that that C-terminal conjugation resulted in the highest contrast. Site specificity is also of great importance for labeling antibodies, as conjugation in the antigen-binding regions might influence the affinity. A method for site-specific labeling of antibodies using an IgG-binding domain that becomes covalently attached to the Fc-region of an antibody by photoconjugation was optimized. By investigation of positions most suitable for incorporation of the photoreactive probe, the conjugation efficiencies were increased for antibody subclasses important for both diagnostic and therapeutic applications. In addition, optimized variants were used in combination with an incorporated click-reactive handle for selective labeling of the antibody with a detection molecule. / <p>QC 20140929</p>
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Effects of antibody labeling chemistry on assays developed for the Gyrolab immunoassay platformDencker, Julia January 2021 (has links)
The aim of this project was to make a comparison of the effects of antibody labeling chemistries on assays developed for the Gyrolab immunoassay platform. One of the labeling techniques was a heterogenous labeling technique targeting amino groups on the antibody. The other labeling technique was a site-specific labeling technique targeting the conserved Fc-glycan at the aspargine 297 residue on the IgG molecule. The site-specific labeling was performed using a kit from Genovis called GlyCLICK. The two labeling techniques were compared on four different assays developed for the Gyrolab platform. The assays tested in this project were two anti-drug antibody assays, a pharmacokinetics assay, a polyclonal antibody assay, and a monoclonal antibody assay. The drug tolerance was tested for the anti-drug antibody assays, resulting in better drug tolerance for reagents labeled with amino conjugation for the Humira assay with incubation overnight. A confirmatory analysis, testing the inhibition of negative control with addition of unlabeled drug in the Master Mix, was performed. This resulted in small differences in the inhibition between the different reagents, except for Keytruda on Gyrolab Bioaffy 200, for which the GlyCLICK labeled reagents led to a lower inhibition of the negative control. For all the assays the effects on signal to background ratio and limit of detection was investigated. The greatest advantages of GlyCLICK on the signal to background was observed for anti-drug antibody Keytruda assay and polyclonal antibody assay. For the polyclonal antibody assay, the results indicated potentially reduced need for the polishing step and for two wash solutions after addition of the detect reagent.
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